Methods for simultaneously detecting both members of a binding pair

ABSTRACT

Methods and kits for simultaneously measuring both members of a binding pair are described.

TECHNICAL FIELD

The invention relates to methods for simultaneously detecting bothmembers of a binding pair in a biological sample.

Background of the Invention

Blood products used for transfusion and transfer of blood componentsmust be routinely screened for the presence of infectious agents such ashuman immunodeficiency virus (HIV), hepatitis viruses, humanT-lymphocytotropic virus, and cytomegalovirus. Such agents typically aredetected by either identification of viral antigens or by detection ofan immune response to the virus (i.e., host-derived antiviralantibodies) using enzyme immunoassay analysis (EIA) or radioimmunoassays(RIA). Immunoassay techniques are limited in their ability to detect thepresence of viral contaminants in early stages of infection, with thewindow period between infection with a virus and detection byimmunoassay techniques varying from two to four weeks for HIV and up toabout 10 weeks for hepatitis C virus (HCV). Techniques such asreverse-transcriptase polymerase chain reaction (RT-PCR) or branchedchain DNA analysis can shorten the time period between infection anddetection, but are cost prohibitive for use on an individual donor basisand do not eliminate the window period.

Summary of the Invention

The invention is based on a rapid and sensitive method forsimultaneously detecting both members of a binding pair, such as aligand and receptor or an antigen and host antibody, from a biologicalsample. Methods of the invention can, for example, enhance the abilityto detect infections at an early stage, leading to earlier treatment ofthe infection.

The invention features a method for simultaneously measuring bothmembers A and B of a binding pair in a biological sample. The biologicalsample is selected from the group consisting of blood, plasma, serum,urine, cerebrospinal fluid, sputum, tears, amniotic fluid, vitreoushumor, saliva, and tissue culture supernatants. The method includesproviding a solid phase reagent, which includes a particle coated withcapture antibodies having specific binding affinities for member A ofthe binding pair, and contacting a biological sample with the solidphase reagent under conditions in which member A, if present, becomesbound to the particle, to form a first reacted particle. The captureantibodies can be monoclonal. The first reacted particle is contactedwith first antibodies having specific binding affinities for member A,wherein the first antibodies are labeled with a first label, and withsecond antibodies having specific binding affinities for member B of thebinding pair, wherein the second antibodies are labeled with a secondlabel, to form a second reacted particle. The first and secondantibodies can be monoclonal. First and second labels (e.g.,fluorophores) are measured on the second reacted particle using flowcytometry.

In certain embodiments, substantially all capture antibodies areoriented on the particle such that the antigen binding regions of thecapture antibodies are available for binding member A of the bindingpair.

Member A of the binding pair can be, for example, an antigen and memberB can be a host antibody. The antigen can be a viral antigen such as ahepatitis C antigen, a hepatitis B antigen, or a human immunodeficiencyvirus antigen, or an autoantigen such as glutarnic acid decarboxylase.Member A of the binding pair also can be a ligand, such as a cytokine,and member B can be a receptor, such as a cytokine receptor. Inaddition, member A can be an enzyme and member B can be a substrate. Forexample, the enzyme can be caspase-3 or caspase-1 and the substrate canbe poly(ADP-ribose) polymerase or proInterleukin-1, respectively.

The invention also features a kit for simultaneously measuring bothembers A and B of a binding pair in a biological sample. The kitincludes a solid phase reagent, which includes a particle coated withcapture antibodies having specific binding affinities for member A ofthe binding pair; first antibodies having specific binding affinitiesfor member A of the binding pair, wherein the first antibodies arelabeled with a first label; and second antibodies having specificbinding affinities for member B of the binding pair, wherein the secondantibodies are labeled with a second label. Substantially all thecapture antibodies are oriented on the particle such that the antigenbinding regions of the capture antibodies are available for bindingmember A of the binding pair. The kit further can include a label orpackage insert, which indicates that the solid phase reagent, thelabeled first antibodies, and the labeled second antibodies can be usedfor simultaneously measuring both members A and B of a binding pair in abiological sample by flow cytometry.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of an assay for detecting member Aand host anti-member A antibody (member B).

FIGS. 2A-2H are scattergrams that indicate simultaneous detection ofhepatitis B virus (HBV) surface antigen, anti-HBV host antibody, HCVcore antigen, and anti-HCV host antibody by flow cytometry.

FIG. 2A is HBV antigen and antibody in a normal sample.

FIG. 2B is HCV antigen and antibody in a normal sample.

FIG. 2C is HBV antigen and antibody in an HBV positive sample.

FIG. 2D is HCV antigen and antibody in an HBV positive sample.

FIG. 2E is HBV antigen and antibody in an HCV positive sample.

FIG. 2F is HCV antigen and antibody in an HCV positive sample.

FIG. 2G is HBV antigen and antibody in an HBV positive/HCV positivesample.

FIG. 2H is HCV antigen and antibody in an HBV positive/HCV positivesample.

DETAILED DESCRIPTION Immunoassay Format

In general, the invention uses a sandwich immunoassay method forsimultaneously detecting both members of a binding pair in a biologicalsample. Binding pairs include any combination of molecules which forms acomplex, including pairs composed of nucleic acids, proteins, or a smallmolecule and a protein. Nucleic acid pairs can be DNA:RNA pairs orDNA:DNA pairs. For example, a DNA/RNA binding pair such as a singlestranded (ss) DNA and an mRNA can be used as a PCR product detectionsystem. A DNA/DNA binding pair such as a ssDNA and a viral DNA can beused in a competition assay for quantitation of virus per amplified DNA.

Non-limiting examples of protein, or small molecule and protein, bindingpairs include a hormone, a cytokine, a peptide, a drug, a viral protein,or other antigen and a cognate receptor or host antibody. Viralprotein/receptor binding pairs can be, for example, HIV gp120 andsoluble CD4. Drug and drug receptor binding pairs can be, for example,cocaine and a dopamine receptor. Peptide and peptide receptor bindingpairs can be, for example, acetylcholine and a muscarinic receptor ordopamine and a dopamine receptor. Hormone and hormone receptor bindingpairs can be, for example, insulin and insulin receptor. Cytokine andcytokine receptor binding pairs can be, for example, tumor necrosisfactor (TNF) and a TNF Type I or Type 2 receptor or interleukin 2 (IL-2)and IL-2 receptor. Antigen and antibody pairs can be, for example, aviral protein and host anti-viral protein antibody or an autoantigen anda host anti-autoantigen antibody. HIV p24/human anti-HIV antibody, HIVgp120/human anti-HIV gp120 antibody, HBV surface antigen/human anti-HBVsurface antigen, and HCV core protein/human anti-HCV core antibody areexamples of viral protein and host antibody binding pairs. Anautoantigen and host anti-autoantigen antibody binding pair can be, forexample, glutamic acid decarboxylase (GAD) and host anti-GAD antibody.

Other protein binding pairs that can be detected are enzyme and enzymesubstrate binding pairs. For example, the enzyme/substrate pair can becaspase-3/poly (ADP-ribose) polymerase or caspase-1/prolnterleukin-1.

Member A, which can be either member of the binding pair, is capturedwith a solid phase reagent that is a particle coated with captureantibodies having specific binding affinities for member A. For example,if the binding pair to be simultaneously detected is HIV gp120/hostanti-HIV gp120 antibody, the particle can be coated with antibodieshaving specific binding affinities for HIV gp120 or anti-hostimmunoglobulin (Ig).

Member A is captured by contacting a biological sample with the particlecoated with capture antibodies. As used herein, suitable biologicalsamples contain cells or cellular material, and include, for example,blood, plasma, serum, urine, saliva, sputum, tears, amniotic fluid,vitreous humor, and cerebrospinal fluid. Other samples can include invitro tissue culture medium/supernatants. Biological samples can betreated with a non-ionic detergent such as 0.5% Triton-X 100 or NonidetP40 (Sigma Chemical Company, St. Louis, Mo.) to expose core antigensfrom pathogens.

The solid phase reagent and biological sample are contacted underconditions that facilitate binding of member A, if present, to theparticle, to form a first reacted particle. Such conditions can includeuse of buffer containing 1% fetal bovine serum (FBS) and 0.1% sodiumazide in phosphate-buffered saline (PBS) at room temperature, or use ofany biological fluid, under physiologic pH conditions. The first reactedparticle then is contacted with two sets of labeled antibodies (i.e.,reporter antibodies) to form a second reacted particle. The firstantibodies have specific binding affinities for member A and are labeledwith a first label. First antibodies are capable of binding to member Awhile member A is bound to capture antibodies. Thus, the captureantibodies and first antibodies must work as a pair. Second antibodieshave specific binding affinities for member B of the binding pair andare labeled with a second label. Fluorescently labeled antibodies areparticularly useful in this method.

FIG. 1 provides a schematic of an assay for detecting member A and hostanti-member A antibody. In this embodiment, biotinylated captureantibodies have specific binding affinities for member A and are coupledto antigen capture beads via avidin. First antibodies have specificbinding affinities for member A and are labeled with phycoerythrin(first label). Second antibodies are labeled with cyanine-phycoerythrin(second label) and have specific binding affinities for host Ig (memberB). The first reacted particle includes member A, host anti-member Aantibodies, capture antibodies, and the solid phase reagent (e.g,antigen capture beads), wherein the second reacted particle includes thefirst reacted particle and the two labeled antibodies.

Flow cytometry can be used to measure the amount of label on the secondreacted particle. As used herein, the term “measure” refers toqualitative and quantitative measurements. In other words, the term“measure” includes reporting the presence or absence of label on thesecond reacted particle, as well as determining the amount of labelpresent. Flow cytometers are able to measure at least three discretefluorescence emission wavelength ranges by using optical filters tosplit the fluorescent emission and separate photomultiplier tubes toamplify the individual emission signals. The intensity of fluorescentemission associated with the particles is directly proportional to theconcentration of analyte present in the biological sample. Thus, the useof different dyes with different emission spectra, wherein each dye iscoupled to a different antibody, allows analysis of multiple analytesper population of particles. The flow cytometer also can distinguishparticles of different sizes such that a particle, for exampleapproximately 7 μm in diameter, can be differentiated from a particleapproximately 10 μm in diameter. Therefore, additional components can bedetected by using a combination of multiple fluorescent dyes and two orthree populations of particles of different average diameters.

Production of Antibodies

Antibodies having specific binding affinities for member A or member Bcan be produced through standard methods. Alternatively, antibodies maybe commercially available, for example, from BiosPacific (Emeryville,Calif.), Coulter (Hialeah, Fla.), Maine Biotechnology Service (Portland,Me.), or Biodesign International (Kennebunk, Me.). As used herein, theterms “antibody” or “antibodies” include intact molecules as well asfragments thereof which are capable of binding to an epitopicdeterminant in member A or member B. The term “epitope” refers to anantigenic determinant on an antigen to which the paratope of an antibodybinds. Epitopic determinants usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chains,and typically have specific three dimensional structuralcharacteristics, as well as specific charge characteristics. Epitopesgenerally have at least five contiguous amino acids. Thus, the terms“antibody” and “antibodies” include polyclonal antibodies, monoclonalantibodies, humanized or chimeric antibodies, single chain Fv antibodyfragments, Fab fragments, and F(ab)₂ fragments. Monoclonal antibodiesare particularly useful.

In general, a protein of interest is produced recombinantly, by chemicalsynthesis, or by purification of the native protein, and then used toimmunize animals. Various host animals including, for example, rabbits,chickens, mice, guinea pigs, and rats, can be immunized by injection ofthe protein of interest. Adjuvants can be used to increase theimmunological response depending on the host species and includeFreund's adjuvant (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin (KLH), and dinitrophenol. Polyclonal antibodies areheterogenous populations of antibody molecules that are specific for aparticular antigen, which are contained in the sera of the immunizedanimals. Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular epitope contained within an antigen, can beprepared using standard hybridoma technology. In particular, monoclonalantibodies can be obtained by any technique that provides for theproduction of antibody molecules by continuous cell lines in culturesuch as described by Kohler, G. et al., Nature, 1975, 256:495, the humanB-cell hybridoma technique (Kosbor et al., Immunology Today, 1983, 4:72;Cole et al., Proc. Natl. Acad. Sci. USA, 1983, 80:2026), and theEBV-hybridoma technique (Cole et al., “Monoclonal Antibodies and CancerTherapy”, Alan R. Liss, Inc., 1983, pp. 77-96). Such antibodies can beof any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and anysubclass thereof. The hybridoma producing the monoclonal antibodies ofthe invention can be cultivated in vitro or in vivo.

A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine monoclonal antibody and a humanimmunoglobulin constant region. Chimeric antibodies can be producedthrough standard techniques.

Antibody fragments that have specific binding affinity for member A or Bcan be generated by known techniques. For example, such fragmentsinclude, but are not limited to, F(ab′)₂ fragments that can be producedby pepsin digestion of the antibody molecule, and Fab fragments that canbe generated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed. See, forexample, Huse et al., 1989, Science, 246:1275. Single chain Fv antibodyfragments are formed by linking the heavy and light chain fragments ofthe Fv region via an amino acid bridge (e.g., 15 to 18 amino acids),resulting in a single chain polypeptide. Single chain Fv antibodyfragments can be produced through standard techniques. See, for example,U.S. Pat. No. 4,946,778.

Once produced, antibodies or fragments thereof are tested forrecognition of member A or member B by standard inmnunoassay methodsincluding, for example, ELISA techniques or RIA. See, Short Protocols inMolecular Biology, Chapter 11, Green Publishing Associates and JohnWiley & Sons, Edited by Ausubel, F.M et al., 1992. Suitable antibodiespreferably have equal binding affinities for recombinant and nativeproteins.

Alternatively, antibodies can be assessed for their ability to formbinding pairs in a fluorescent sandwich assay in the following manner.Beads can be coated with biotinylated antibodies, for example anti-viralprotein antibodies, then incubated for approximately 30 minutes with 2ng/ml of the appropriate protein, e.g., recombinant viral protein, in a100 μl volume. After washing the beads twice with 2 ml of buffercontaining 1% FBS and 0.1% sodium azide in PBS, the beads are incubatedwith approximately 0.5 μg of phycoerythrin-labeled antibody. Pairs ofantibodies producing a strong fluorescent signal are suitable for use inassays of the invention.

Solid Phase Reagents

Suitable particles (e.g, beads) have an average diameter of about 2 μmto 15 μm and can be polystyrene, ferromagnetic, or paramagnetic. Forexample, the particles can have an average diameter of about 4 μm toabout 11 μm. Typical average particle diameters are about 4-5 μm, 7-8μm, and 10-11 μm. Particles are available commercially, for example,from Spherotech Inc., Libertyville, Ill. Particles can be coated withcapture antibodies by known techniques. For example, avidin- orstreptavidin-coated paramagnetic beads can be coated with biotinylatedcapture antibodies. In general, avidin- or streptavidin-coated beads areresuspended in a saline solution, such as PBS, mixed with biotinylatedantibodies at saturating conditions (approximately 40 μg of protein per3.9×10⁷ 7 μm beads), and incubated at room temperature. After binding iscomplete, the beads are washed and blocked with, for example, buffercontaining 1% FBS and 0.1% sodium azide in PBS.

Avidin- or streptavidin-coated beads can be coupled to biotinylatednucleic acids when nucleic acid binding pairs are being measured.Nucleic acids can be labeled with biotin by incorporation ofbiotin-11-dUTP in a standard nick translation reactions.

In particular embodiments, substantially all of the capture antibodiesare oriented on the particle such that the antigen binding regions areavailable for binding member A, increasing overall sensitivity of theassay. The term “substantially all” indicates that at least 80%, andpreferably at least 90%, (e.g., 95% or 99%) of the antibodies areoriented in this fashion. Percent orientation can be estimatedqualitatively by measuring fluorescence associated with binding ofphycoerythrin-labeled goat anti-mouse antibody, and comparing withstandardized fluorescent particles. Antigen binding regions ofantibodies are available for binding member A when the antibody isbiotinylated at amino acid residues primarily outside of the antigenbinding region. Thus, during biotinylation of antibodies, a molar ratioof biotin:antibody of about 5:1 to about 10:1 and other standardreaction conditions are used. For example, biotin N-hydroxysuccinimidylester or biotin succinimidyl ester can be used at a pH of about 8.1.Alternatively, biotin hydrazide can be used at a pH of 4.5-5.0.

Assay sensitivity also is increased because capture of member A from abiological sample is not limited to reaction volumes of 200 μl or less,as in traditional assays. Particles are easy to collect from largevolumes of biological sample by either magnetic separation orcentrifugation. Furthermore, each particle contains, on average,approximately 180,000 to 240,000 antibody binding sites, andapproximately 300,000 to 350,000 biotinylated antigen binding sites perparticle. Thus, each particle has a large binding capacity and a largeeffective range of analysis for antigen concentration.

Detectable Labels

Each labeled antibody can be distinctly visualized by labeling with afluorophore that emits light of a color that contrasts with otherfluorophores. For example, a combination of the following fluorophoresmay be used: 7-amino-4-methylcoumarin-3-acetic acid (AMCA), Texas Red™(Molecular Probes, Inc., Eugene, OR), 5-(and-6)-carboxy-X-rhodamine,lissamine rhodamine B, 5-(and-6)-carboxyfluorescein,fluorescein-5-isothiocyanate (FITC), 7-diethylaminocoumarin-3carboxylicacid, tetramethylrhodamine-5-(and-6)-isothiocyanate,5-(and-6)-carboxytetramethylrhodamine, 7-hydroxycoumarin-3-carboxylicacid, 6-[fluorescein 5(and-6)-carboxamido]hexanoic acid,N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a diaza-3-indacenepropionicacid, eosin-5-isothiocyanate, erythrosin-5-isothiocyanate, phycoerythrin(B-, R-, or cyanine-), allophycocyanin, Oregon Green™, and Cascade™ blueacetylazide (Molecular Probes, Inc., Eugene, Oreg.).

Antibodies also can be labeled with semiconductor nanocrystals. Watersoluble nanocrystals are composed of different sizes ofcadmium-selenium/cadmiumsulfur core-shell nanocrystals enclosed in asilica shell or cadmium-selenium/zincsulfur nanocrystals solubilized inmercaptoacetic acid. Such water soluble nanocrystals have a narrow,tunable, symmetric emission spectrum and are photometrically stable.See, Bruchez Jr. et al., Science, 1998, 281:2013-2016; and Chan et al.,Science, 1998, 281:2016-2018.

Detection of Multiple Antigens and Host Antibody

A combination of labels, such as Oregon Green™ (Molecular Probes, Inc.,Eugene, Oreg.), phycoerythrin, and cyanine-phycoerythrin, can be used todetect, inter alia, two antigens and a host antibody. For example, HCV,HBV surface antigen, host anti-HCV antibody, and host anti-HBV surfaceantigen antibody can be simultaneously detected usingphycoerythrin-labeled antibodies having specific binding affinities forHCV, Oregon Green™-labeled antibodies having specific binding affinitiesfor HBV surface antigen, and cyanine-phycoerythrin-labeled anti-host Igantibodies. Using three different labels and two populations ofparticles having different sizes (e.g., average diameters of 7-8 μm and10-11 μm) allows up to 6 different viral antigens and host antibodies tobe detected simultaneously. Use of a third population of particles of adifferent average diameter allows up to 9 different viral antigens andhost antibodies to be detected.

Viral antigens can be difficult to detect in plasma samples once anindividual has seroconverted (i.e, has developed host antibodies)because the binding sites for the capture or reporter antibodies on theviral particle have been blocked by the host antibody. The presentinvention overcomes this difficulty due to the improved sensitivity ofthe assay over traditional immunoassay formats. Thus, viral antigen canbe detected using the present methods in situations in which traditionalimmunoassay formats cannot do so. As described herein, viral antigenscan be captured using particles coated with monoclonal antibodies havingspecific binding affinities for the viral protein, and their presencedetected with reporter monoclonal antibodies directed against the viralprotein in seropositive individuals. Host antibody directed against theviral protein can be simultaneously detected through labeled goatanti-human Ig. Detection of viral protein without host antibodyindicates the host was recently infected and has not seroconverted,while detection of viral protein and host antibody indicates thepresence of infection as well as seroconversion. In certain samples,host antibody may be detected but viral protein is not when, forexample, binding sites for the first antibody are not available.Although viral proteins are not directly measured in this instance,viral proteins are still present in the sample, as the antibody-coatedcapture bead directed against the viral protein captures the immunecomplex of viral antigen and host antibody.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Biotinylation of Proteins

Antibodies were conjugated at a concentration of 5 mg/ml; viral antigenswere conjugated at a concentration of 1 mg/ml. To biotin labelanti-viral protein antibodies and viral antigens, the proteins wereexchanged into 100 mM KH₂CO₃ buffer (pH 8.3) using an appropriate sizeCentricon (Amicon) filter.

Biotin N-hydroxysuccinimidyl ester (Molecular Probes, Eugene, Oreg.) inDMSO (10 mg/ml, Sigma Chemical Co., St. Louis, Mo., Cat. #D8779) wasprepared immediately prior to use, and added to the protein to bebiotinylated in a 5:1 or 10:1 molar ratio. Reactions were performed byvortexing the protein solution lightly, and adding the biotin/DMSO tothe protein solution and mixing thoroughly. Protein and biotin esterwere reacted for one hour at room temperature in the dark. Conjugatedprotein was separated from free biotin by separation on a 10 mlSephadex-25 gel column or spin column using 1×PBS to elute. Individual 1ml fractions were collected and absorbance at A280 nm was measured.Fractions representing the initial peak of A280 were collected andpooled, while remaining fractions, including those representing thesecond A280 peak, were discarded.

When spin columns were used, the reaction mixture was distributedequally between four spin columns, and spun using a Serofage centrifugeon high speed for 2 mins. Material passing through the column wascollected and the columns were washed by filling the column with 1×PBSand spinning at high speed in a Serofuge for 2 min and repeating fivetimes. Collected material was redistributed equally among four columns,then spun using the Serofage centrifuge on high speed for 2 min.Material passing through the column was collected, pooled, andre-analyzed for A280 and the concentration was determined. Conjugatedprotein was stored at 4° C.

Example 2 Production of Analyte Capture Beads

Analyte capture beads were prepared by completely resuspendingavidin-coated paramagnetic beads (7 μm, Spherotech, VM-60-100) by mixingwell. Beads (typically 3.8×10⁶ beads) were placed in a 50 ml centrifugetube and mixed with 30 ml of 1×PBS. After retaining beads on the side ofthe tube with magnets, all PBS was removed. The beads were washed twomore times with PBS.

After the final PBS wash, the required volume of biotinylated antibody(typically 40 μg) and 2 ml of 1×PBS were added to the beads. The beadswere resuspended by vortexing continuously for a minimum of 3 hours, orby vortexing for one hour and then storing overnight at 4° C. Beadsstored overnight were vortexed for an additional 2 hours the nextmorning. Approximately 30 ml of buffer containing 1% FBS and 0.1% NaN₃in PBS were used to wash the conjugated beads three times. Beads wereresuspended in 19.25 ml of the same buffer and stored at 4° C. untiluse.

To label antibodies with the fluorescein derivative Oregon Green™(Molecular Probes, Eugene, Oreg.), antibodies were exchanged into 100 mMKH₂CO₃ buffer (pH 9.0) at a concentration of 5 mg/ml. Oregon Green™ (10mg/ml in dimethylformamide, DMF) was added to the antibody at a 25:1molar ratio and incubated for 1 hour at room temperature, in the dark.Free Oregon Green™ was separated from the antibody on a G-25 Sephadexcolumn. R-phycoerythrin (PE, Intergen BioDiagnostics, Purchase, N.Y.)and cyanine-phycoerythrin (CySPE) conjugates were produced using2-iminothiolane (Pierce Chemical Co., Rockford, Ill.) in a 1625:1 molarratio to modify the fluorochrome and sulfo-SMCC (Pierce) in a 20:1 molarratio to modify the antibody. Modified fluorochrome and antibody wereincubated together for 1 hour at room temperature in the dark. Freefluorochrome and antibody were separated from fluorochrome-conjugatedantibody on Sephacryl S-300-HR columns (Sigma Chemical Co., St. Louis,Mo.). Goat F(ab′)₂ anti-human Ig antiserum (heavy and light chainspecific) affinity-purified and absorbed against mouse, equine, bovine,rat, and rabbit antibodies was labeled with PE or Cy5-PE. Alterations inthe ratio of fluorochrome to protein can be made to optimize thefluorescent signal for a particular antibody or viral antigen.

Example 3 Detection of Viral Antigens and Host Antibody

Plasma samples from normal individuals and from individuals positive forHCV, HIV, or HBV were obtained from New York Biologicals (Southampton,N.Y.), Scantibodies Laboratory (Santee, Calif.), or IntergenBioDiagnostics (Purchase, N.Y.). Plasma samples were treated withTriton-X 100 detergent to a final concentration of 0.5% to lyse viralmembranes and expose core particles prior to testing. E. coli derivedrecombinant viral antigens, including surface and core antigens, wereobtained from BiosPacific (Emeryville, Calif.) or IntergenBioDiagnostics. Antigens were added to normal nonpathologic serumsamples for development of a reference standard curve and for use inspike and recovery analysis.

Flow cytometric analysis was performed on a Coulter EPICS Profile II, aCoulter XL, or a Partec PAS flow cytometer using linear forward vs. sidelight scatter to gate the bead population. Fluorescence signal wasamplified logarithmically. Fluorescence emissions were segregated intodiscrete colors by optical filters. A 525 nm bandpass filter was used tocollect the green fluorescence (Oregon Green and FITC), a 565 nmbandpass filter to collect the orange fluorescence (PE), and a 630 nmlong pass filter to collect the red fluorescence (Cy5 PE).

Samples were incubated with PE- and Cy5PE-labeled F(ab′)₂ goatanti-human Ig (heavy and light chain specific) antibody. In each case,host anti-viral antibodies were detected on beads with captured antigenand not on beads from normal samples or an incorrect virus. BBV surfaceantigen and anti-HBV antibody, as well as HCV core antigen and anti-HCVantibody, were detected using a PE labeled antibody having specificbinding affinity for HCV core antigen, an Oregon Green labeled antibodyhaving specific binding affinity for HBV surface antigen, and a CySPElabeled goat anti-human Ig antibody. As indicated in FIGS. 2A-2H,individual and simultaneous detection of HCV, HBV, and host antibodywere possible.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for simultaneously measuring bothmembers A and B in a binding pair complex in a biological sample, saidmethod comprising: a) providing a solid phase reagent, said solid phasereagent comprising a particle coated with capture antibodies havingspecific binding affinities for said member A of said binding paircomplex; b) contacting said biological sample with said solid phasereagent under conditions in which said member A, if present, becomesbound to said particle, to form a first reacted particle: c) contactingsaid first reacted particle with first antibodies having specificbinding affinities for said member A, wherein said first antibodies arelabeled with a first label, and with second antibodies having specificbinding affinities for said member B of said binding pair complex,wherein said second antibodies are labeled with a second label, to forma second reacted particle, wherein said first and second labels aredifferent and d) measuring said first and second labels on said secondreacted particle using flow cytometry.
 2. The method of claim 1, whereinsubstantially all said capture antibodies are oriented on said particlesuch that the antigen binding regions of said capture antibodies areavailable for binding said member A of said binding pair complex.
 3. Themethod of claim 1, wherein said member A is an antigen and said member Bis a host antibody.
 4. The method of claim 3, wherein said antigen is aviral antigen.
 5. The method of claim 4, wherein said viral antigen is ahepatitis C antigen.
 6. The method of claim 4, wherein said viralantigen is a hepatitis B antigen.
 7. The method of claim 4, wherein saidviral antigen is a human immunodeficiency virus antigen.
 8. The methodof claim 1, wherein said antigen is an autoantigen.
 9. The method ofclaim 8, wherein said autoantigen is glutamic acid decarboxylase. 10.The method of claim 1, wherein said member A is a ligand and said memberB is a receptor.
 11. The method of claim 10, wherein said ligand is acytokine and said receptor is a cytokine receptor.
 12. The method ofclaim 1, wherein said member A is an enzyme and said member B is asubstrate.
 13. The method of claim 12, wherein said enzyme is caspase-3and said substrate is poly(ADP-ribose) polymerase.
 14. The method ofclaim 12, wherein said enzyme is caspase-1 and said substrate isproInterleukin-1.
 15. The method of claim 1, wherein said first andsecond labels are fluorophores.
 16. The method of claim 1, wherein saidbiological sample is selected from the group consisting of blood,plasma, serum, urine, cerebrospinal fluid, sputum, tears, amnioticfluid, vitreous humor, saliva, and tissue culture supernatants.
 17. Themethod of claim 1, wherein said capture antibodies are monoclonal. 18.The method of claim 1, wherein said first antibodies are monoclonal. 19.The method of claim 1, wherein said second antibodies are monoclonal.20. A kit for simultaneously measuring both members A and B in a bindingpair complex in a biological sample, said kit comprising: a) a solidphase reagent, said solid phase reagent comprising a particle coatedwith capture antibodies having specific binding affinities for saidmember A of said binding pair complex, wherein substantially all saidcapture antibodies are oriented on said particle such that the antigenbinding regions of said capture antibodies are available for bindingsaid member A of said binding pair complex; b) first antibodies havingspecific binding affinities for said member A of said binding paircomplex, wherein said first antibodies are labeled with a first label;and c) second antibodies having specific binding affinities for saidmember B of said binding pair complex, wherein said second antibodiesare labeled with a second label, and wherein said first and secondlabels are different.